Fluorescence polarization and fluorescence intensity measurements provide a powerful means by which macromolecular association reactions can be studied. These fluorescent techniques have been applied to study antigen-antibody, hapten-antihapten, protein-ligand, and protein-DNA interactions.
The inherent sensitivity of fluorescence measurements can be used in monitoring the extent of reaction as a fluorescent reactant, F, combines with its macromolecular partner, R:
where k1 is the forward reaction and k−1 is the back reaction such that (k1)/(k−1)=K(eq).
The investigator can choose to follow changes in the fluorescence polarization (FP) and/or the fluorescence intensity (FI). If the reactants do not have natural fluorescence, as in the case of many antigen-antibody systems, one of the reactants can be covalently labeled with a fluorescent tag. An increase in the fluorescence polarization of F usually occurs during combination with R, even if there are no concomitant changes in the fluorescence intensity. This is because the polarization increase reflects a slowing down of the rotary motion of the smaller ligand, F, when it becomes attached to the larger species, R. R is in many instances an antibody or a fragment of an antibody, such as an Fab or Fab2 (dimer). Equilibrium fluorescence polarization and intensity measurements can be determined in a direct readout polarometer capable of measuring both the degree of fluorescence polarization and the fluorescence intensity of a solution.
Immunoassays have been used in an effort to improve upon the success in detecting substances at very low levels. For example, the use of such techniques has been prompted by the extraordinary successes that have been achieved in the measurement of biological substances by specific immunological reagents and techniques. Available evidence indicates that specific antibodies can be obtained against even low molecular weight organic compounds, such as pesticides or other haptens.
Any means of applying an immunochemical reaction to a detection problem ultimately relies upon a reaction occurring between a substance (antigen or hapten) and its specific antibody. One means by which this interaction can be employed in measurement and detection has come to be known as “competitive binding assay”. In principle, this method requires two reagents. These are a labeled form of the substance to be detected or measured, and an antibody or receptor specifically directed against the substance. The principle of the assay involves a preliminary measurement of the binding of the labeled hapten or antigen (substance being detected) with its antibody and then, a determination of the extent of the inhibition of this binding by known quantities of the unlabeled hapten or antigen, which corresponds to the unknown. From these data, a standard curve can be constructed which shows the degree of binding by the labeled hapten or antigen under certain specified conditions as a function of concentration of the unlabeled hapten or antigen or unknown added.
One way of implementing an immunoassay is to employ a fluorescent label. Usually, fluorescent labeling of one of the reagents e.g. the hapten is important in carrying out of the immunoassay by means of fluorescence polarization and/or fluorescence intensity measurements. Unlike other immunoassays, such as ELISA, no physical separation of bound from free forms of the labeled hapten is necessary. Therefore a simple rapid optical measurement yields the essential information without physical separation of bound and free labeled materials.
One problem associated with immunoassays, as well as other assays, has been non-specific binding. Ideally, in an immunoassay, the investigator wants to follow a simple biomolecular reaction occurring between a labeled substance (antigen or hapten) and its specific antibody. However, the investigator must often contend with non-specific binding, such as antibody-antibody interactions, antigen-antigen interaction, hapten-hapten interaction, or interactions between the antibody or antigen (or hapten) and interfering substances in the assay. Such non-specific binding can make it extremely difficult, if not impossible to measure specific binding reactions, especially when the equilibrium and rate constants for non-specific binding reactions are a significant fraction of those of the specific binding reactions.
Therefore, there is a need to provide improved and more sensitive assays for detecting the presence and/or amount of an analyte in a sample. In particular, it would be advantageous to provide competitive and non-competitive assays in which non-specific binding in the assays is substantially reduced. This would allow, for example, the investigator to successfully follow the specific binding of a labeled antigen or hapten (substance being detected) with its antibody, an unlabeled antibody with a tracer, an unlabeled analyte with a labeled antibody, and many other analytes or labeled tracers with an appropriate “binding partner”, such as a fragment of an antibody, a receptor, or other proteins.